丙戊酸钠联合雌激素对去卵巢痴呆小鼠的作用及其机制的初步研究
发布时间：2018-08-27 09:57
【摘要】：目的:研究雌激素缺乏不同时间段痴呆模型小鼠脑内雌激素受体(Estrogen receptors,ERs)及糖原合成酶激酶3β(glycogen synthase kinase 3β)的动态变化,并观察丙戊酸钠(valproic acid sodium salt,VPA)联合雌激素对APP/PS1双转基因去势AD模型小鼠脑内雌激素受体含量及GSK-3β相关信号通路的影响,探讨相关的作用机制。本课题研究成果将为进一步揭示AD的发病机制及临床上研究和开发治疗AD的多靶向药物提供新的实验室依据。方法:本课题分两部分进行。第一部分:选取3月龄雌性APP/PS1双转基因小鼠40只,将其随机分为假手术组(AD-Sham组)和手术组(AD-OVX组),同月龄野生型小鼠为对照组(WT组),每组10只。分别于术后1个月和术后5个月,采用多种方法检测小鼠外周血雌激素水平、行为学、脑组织形态学及相关蛋白的变化。第二部分:选取去势后1个月痴呆模型小鼠进行药物治疗,随机平均分为6组:生理盐水对照组、VPA药物处理组、E2药物处理组、Liq药物处理组、VPA联合E2药物处理组及VPA联合Liq药物处理组,每组8只。vpa药物处理组小鼠给予药物vpa30mg/kg·d,雌激素药物处理组给予e2药物2.4ug/只·d,liq治疗组给予liq50ug/kg·d,vpa和e2联合治疗组给予vpa30mg/kg·d和e22.4ug/只·d,vpa和liq联合治疗组给予vpa30mg/kg·d和liq50ug/kg·d,生理盐水治疗组给予等量生理盐水,除liq给药方式为灌胃外,其余给药均为腹腔注射。药物处理1个月后,采用高架十字迷宫及旷场实验以检测各组小鼠的抑郁焦虑行为;morris水迷宫检测小鼠的空间学习及记忆能力;利用蛋白印迹(westernblot)等技术,检测联合用药对小鼠脑内雌激素受体含量,gsk-3β活性及老年斑的影响,并探讨其相关机制。结果:1.第一部分实验结果:ovx1个月和5个月组ad小鼠体循环雌激素水平显著下调(p0.05);ovx1个月组小鼠子宫无明显缩小,而ovx5个月出现明显萎缩。morris水迷宫显示,ovx1个月后,与ad-sham组相比,ad-ovx组小鼠找到平台的逃避潜伏期和路程显著增加(p0.05),跨越平台的次数明显减小(p0.05)。随着ovx时间的延长,至ovx5个月后,ad-ovx组小鼠找到平台的时间和经过的路程更进一步延长(p0.05),跨越平台的次数也相应下降(p0.05)。免疫荧光结果显示,与ad-sham组相比,ad-ovx1个月和5个月组小鼠脑内老年斑均显著增加(p0.05)。westernblot结果显示,与ad-sham组相比,ad-ovx1个月及5个月组小鼠脑内erβ蛋白水平出现进行性下调(p0.05);相应地,ad-ovx组小鼠脑内p-gsk-3β水平降低的趋势与erβ一致(p0.05),尽管正常小鼠和ad小鼠脑内gsk-3β总蛋白无明显差异(p0.05)。2.第二部分实验结果:1、行为学1)高架十字迷宫检测结果显示:联合药物处理组小鼠在开放臂活动次数明显增多(p0.05),联合药物处理组在开放臂停留的时间与其余各组相比均明显延长(p0.01)。在开放臂活动次数及开放臂停留时间的比率上,vpa与e2联合处理组均明显增加(p0.01),liq处理组也明显增加(p0.05);vpa与e2,vpa与liq联合处理组较vpa及e2单独药物处理组也显著增加(p0.05)。2)水迷宫实验结果:可视平台条件下,各组药物处理小鼠找到平台的时间和路程均无明显的差异(p0.05)。隐蔽平台实验条件下,其他各药物处理组小鼠找到隐蔽平台的游动时间及路程与生理盐水对照组相比均明显缩短(p0.05);而联合药物处理组游动时间及距离更加缩短(p0.05)。空间探索实验中,与生理盐水组比较,联合药物处理组穿越平台的次数明显增多(p0.01),单一药物处理组也明显增多(p0.05);与联合药物处理组比较,vpa处理组和liq处理组均明显减少(p0.05),e2处理组显著减少(p0.01)。2、各组小鼠子宫形态及重量统计结果显示:各药物处理组小鼠子宫在外观形态上均有不同程度的增大,统计分析结果得出,vpa组,liq组与control组相比,子宫重量无明显变化(p0.05);e2组,vpa+e2组,vpa+liq与control组相比,子宫重量显著增加(p0.01)。e2组,vpa+e2组小鼠子宫形态与其余各组相比明显增生,重量也显著增加(p0.05),并有大小形状不一的结节形成。3.蛋白水平实验结果:(1)雌激素受体α与β与生理盐水组比较,大脑皮质区erα蛋白含量在联合药物处理组中显著上升(p0.01),在各单独药物处理组中无明显差异(p0.05)。与生理盐水对照组相比,大脑海马区erα蛋白水平在vpa药物处理组中无明显变化(p0.05),在其余各药物处理组中均显著增多(p0.05)。在erβ含量的比较中,与生理盐水对照组相比,其余各药物处理组大脑皮质及海马区内erβ含量均显著增多(p0.05),且联合药物处理组大脑皮质及海马区内erβ的含量比各药物单独处理组明显增多(p0.05)。(2)gsk-3β活性检测:与生理盐水对照组相比,其余各单独药物处理组小鼠脑皮质及海马区gsk-3β总蛋白水平均无明显差异(p0.05),联合药物处理组脑皮质区gsk-3β总蛋白水平显著下降(p0.05),而脑海马区内gsk-3β总蛋白水平无明显差异(p0.05)。其第9位点磷酸化水平标志着gsk-3β活性的高低,与生理盐水对照组相比,vpa组、e2组脑皮质区pser9-gsk-3β水平均无明显差异(p0.05),liq组及联合药物处理组脑皮质区pser9-gsk-3β水平明显升高(p0.05);而在各组脑海马区pser9-gsk-3β水平的比较中,与生理盐水对照组相比,其余各药物处理组pser9-gsk-3β水平均明显升高(p0.05),且与各单独药物处理组相比,联合药物处理组pser9-gsk-3β水平也显著升高(p0.05)。结论:1、卵巢切除手术后,ad小鼠体循环雌激素水平随年龄增长呈下降趋势,子宫形态随年龄增长逐渐萎缩。2、卵巢切除手术后,AD小鼠空间学习记忆能力随年龄增长逐渐下降。3、卵巢切除手术后,AD小鼠脑内Aβ的沉积及老年斑的形成增多,其可能机制可能是与ERβ含量下降所导致的GSK-3β活性异常有关。4、药物处理明显改善了去势AD小鼠的抑郁焦虑行为,缓解了其空间学习记忆能力的下降,且联合用药的效果明显优于单一药物处理。5、药物处理显著增加了去势AD小鼠脑皮质及海马区雌激素受体的含量,且联合用药的效果更显著。6、药物处理显著增加了去势AD小鼠脑皮质及海马区p ser9-GSK-3β的含量,使GSK-3β的活性受到了抑制,且联合用药的效果更显著。
[Abstract]:AIM: To study the dynamic changes of estrogen receptors (ERs) and glycogen synthase kinase 3 beta (GSK 3 beta) in the brain of mice with estrogen deficiency at different time points, and to observe the effect of valproic acid sodium salt (VPA) combined with estrogen on the estrogen in the brain of APP / PS1 castrated AD model mice. The results of this study will provide a new laboratory basis for further revealing the pathogenesis of AD and clinical research and development of multi-targeted drugs for AD. METHODS: This study was carried out in two parts. Part I: Selection of 3-month-old female APP/PS1. Forty double transgenic mice were randomly divided into sham operation group (AD-Sham group) and operation group (AD-OVX group) and wild type mice of the same age group (WT group) with 10 mice in each group. Part: One month after ovariectomy, dementia model mice were randomly divided into 6 groups: normal saline control group, VPA drug treatment group, E2 drug treatment group, Liq drug treatment group, VPA combined with E2 drug treatment group and VPA combined with Liq drug treatment group, with 8 mice in each group. The treatment group was given E2 drug 2.4ug/kg/d, the liq treatment group was given liq50ug/kg/d, the VPA and E2 combined treatment group were given VPA 30mg/kg/d and e22.4ug/kg/d, the VPA and liq combined treatment group were given VPA 30mg/kg/d and liq50ug/kg/d, and the saline treatment group was given the same amount of saline, except the liq was given intraperitoneally. After one month of treatment, the depression and anxiety behaviors of mice in each group were tested by elevated cross maze and open field test; the spatial learning and memory abilities of mice were tested by Morris water maze; the estrogen receptor content, GSK-3 beta activity and senile plaque were detected by Western blot and other techniques. Results: 1. the results of the first part of the experiment: OVX 1 month and 5 months group AD mice circulatory estrogen levels significantly decreased (p0.05); OVX 1 month group mice uterus did not significantly shrink, but OVX 5 months showed significant atrophy. Morris water maze showed that OVX 1 month later, compared with ad-sham group, ad-ovx mice found a platform escape potential. The incubation period and distance increased significantly (p0.05), and the number of crossing platforms decreased significantly (p0.05). with the prolongation of OVX time, to OVX 5 months later, the time of finding platforms and the distance of crossing platforms in ad-ovx group were further prolonged (p0.05), and the number of crossing platforms decreased accordingly (p0.05). immunofluorescence results showed that compared with ad-sham group, ad-ovx 1. The results of Western blot showed that compared with ad-sham group, the level of ER-beta protein in the brain of ad-ovx mice decreased progressively (p0.05); accordingly, the decrease of p-gsk-3 beta level in the brain of ad-ovx mice was consistent with that of normal mice and AD mice (p0.05). There was no significant difference in the total protein of GSK-3 beta between the two groups (p0.05). 2. Part 2: 1, Behavior 1) The results of the elevated maze test showed that the number of activities in the open arm of the mice in the combined drug treatment group was significantly increased (p0.05), and the duration of stay in the open arm of the combined drug treatment group was significantly longer than that of the other groups (p0.01). The ratio of activity times and residence time of open arms in the VPA and E2 combined treatment group were significantly increased (p0.01), and the liq treatment group was also significantly increased (p0.05); VPA and e2, VPA and liq combined treatment group compared with the VPA and E2 single drug treatment group were significantly increased (p0.05). There was no significant difference in the time and distance between the two groups (p0.05). under the covert platform experiment, the swimming time and distance of the mice in the other drug treatment groups were significantly shorter than those in the normal saline control group (p0.05), while the swimming time and distance of the combined drug treatment group were shorter (p0.05). Compared with the saline group, the number of crossing platform in the combined drug treatment group was significantly increased (p0.01), and the number of crossing platform in the single drug treatment group was also significantly increased (p0.05); compared with the combined drug treatment group, VPA treatment group and liq treatment group were significantly reduced (p0.05), E2 treatment group was significantly reduced (p0.01). uterine morphology and weight statistics of mice in each group showed that: The uterus morphology of the mice in the treatment group increased in different degrees. the results of statistical analysis showed that the weight of uterus in VPA group, liq group and control group had no significant change (p0.05); the weight of uterus in E2 group, VPA + E2 group, VPA + liq group and control group increased significantly (p0.01). The results of protein level experiment: (1) compared with the normal saline and estrogen receptor alpha group, the content of ER alpha protein in the cerebral cortex increased significantly in the combined drug treatment group (p0.01), and there was no significant difference in the single drug treatment group (p0.05). Compared with the control group, the level of ER-alpha protein in the cerebral hippocampus did not change significantly in the VPA treatment group (p0.05), but increased significantly in the other drug treatment groups (p0.05). in the comparison of ER-beta content, compared with the normal saline control group, the content of ER-beta in the cerebral cortex and hippocampus of the other drug treatment groups increased significantly (p0.05). (2) GSK-3 beta activity test: compared with the normal saline control group, there was no significant difference in the levels of GSK-3 beta total protein in the cerebral cortex and hippocampus of mice treated with other drugs alone (p0.05), and there was no significant difference in GSK-3 beta total protein between the combined drug treatment group and the normal saline control group (p0.05). The total protein level decreased significantly (p0.05), but there was no significant difference in the total protein level of GSK-3 beta in the hippocampus (p0.05). the phosphorylation level of the ninth locus marked the activity of GSK-3 beta. compared with the normal saline control group, the levels of pser9-gsk-3 beta in the VPA group, E2 group and liq group had no significant difference (p0.05), and the combined drug treatment group. The levels of pser9-gsk-3 beta in the hippocampus of each group were significantly higher than those in the normal saline group (p0.05). The levels of pser9-gsk-3 beta in the other drug treatment groups were significantly higher than those in the control group (p0.05), and the levels of pser9-gsk-3 beta in the combined drug treatment group were also significantly higher than those in the single drug treatment group (p0.05). Conclusion: 1. After ovariectomy, the estrogen level in the systemic circulation of AD mice decreased with age, and the uterine morphology gradually atrophied with age. 2. After ovariectomy, the spatial learning and memory ability of AD mice gradually decreased with age. 3. After ovariectomy, the deposition of A beta in the brain and the formation of senile plaque of AD mice increased, which may be related to ovariectomy. The mechanism may be related to the abnormal GSK-3 beta activity caused by the decrease of ER beta content. 4. Drug treatment significantly improved the depressed and anxious behavior of ovariectomized AD mice, alleviated the decline of their spatial learning and memory ability, and the effect of combined treatment was significantly better than that of single drug treatment. 5. Drug treatment significantly increased the cortex and sea of ovariectomized AD mice. The content of estrogen receptor in horse area was significantly increased. 6. Drug treatment significantly increased the content of P ser9-GSK-3 beta in cerebral cortex and hippocampus of ovariectomized AD mice, and inhibited the activity of GSK-3 beta, and the effect of combined treatment was more significant.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16
本文编号：2206934
[Abstract]:AIM: To study the dynamic changes of estrogen receptors (ERs) and glycogen synthase kinase 3 beta (GSK 3 beta) in the brain of mice with estrogen deficiency at different time points, and to observe the effect of valproic acid sodium salt (VPA) combined with estrogen on the estrogen in the brain of APP / PS1 castrated AD model mice. The results of this study will provide a new laboratory basis for further revealing the pathogenesis of AD and clinical research and development of multi-targeted drugs for AD. METHODS: This study was carried out in two parts. Part I: Selection of 3-month-old female APP/PS1. Forty double transgenic mice were randomly divided into sham operation group (AD-Sham group) and operation group (AD-OVX group) and wild type mice of the same age group (WT group) with 10 mice in each group. Part: One month after ovariectomy, dementia model mice were randomly divided into 6 groups: normal saline control group, VPA drug treatment group, E2 drug treatment group, Liq drug treatment group, VPA combined with E2 drug treatment group and VPA combined with Liq drug treatment group, with 8 mice in each group. The treatment group was given E2 drug 2.4ug/kg/d, the liq treatment group was given liq50ug/kg/d, the VPA and E2 combined treatment group were given VPA 30mg/kg/d and e22.4ug/kg/d, the VPA and liq combined treatment group were given VPA 30mg/kg/d and liq50ug/kg/d, and the saline treatment group was given the same amount of saline, except the liq was given intraperitoneally. After one month of treatment, the depression and anxiety behaviors of mice in each group were tested by elevated cross maze and open field test; the spatial learning and memory abilities of mice were tested by Morris water maze; the estrogen receptor content, GSK-3 beta activity and senile plaque were detected by Western blot and other techniques. Results: 1. the results of the first part of the experiment: OVX 1 month and 5 months group AD mice circulatory estrogen levels significantly decreased (p0.05); OVX 1 month group mice uterus did not significantly shrink, but OVX 5 months showed significant atrophy. Morris water maze showed that OVX 1 month later, compared with ad-sham group, ad-ovx mice found a platform escape potential. The incubation period and distance increased significantly (p0.05), and the number of crossing platforms decreased significantly (p0.05). with the prolongation of OVX time, to OVX 5 months later, the time of finding platforms and the distance of crossing platforms in ad-ovx group were further prolonged (p0.05), and the number of crossing platforms decreased accordingly (p0.05). immunofluorescence results showed that compared with ad-sham group, ad-ovx 1. The results of Western blot showed that compared with ad-sham group, the level of ER-beta protein in the brain of ad-ovx mice decreased progressively (p0.05); accordingly, the decrease of p-gsk-3 beta level in the brain of ad-ovx mice was consistent with that of normal mice and AD mice (p0.05). There was no significant difference in the total protein of GSK-3 beta between the two groups (p0.05). 2. Part 2: 1, Behavior 1) The results of the elevated maze test showed that the number of activities in the open arm of the mice in the combined drug treatment group was significantly increased (p0.05), and the duration of stay in the open arm of the combined drug treatment group was significantly longer than that of the other groups (p0.01). The ratio of activity times and residence time of open arms in the VPA and E2 combined treatment group were significantly increased (p0.01), and the liq treatment group was also significantly increased (p0.05); VPA and e2, VPA and liq combined treatment group compared with the VPA and E2 single drug treatment group were significantly increased (p0.05). There was no significant difference in the time and distance between the two groups (p0.05). under the covert platform experiment, the swimming time and distance of the mice in the other drug treatment groups were significantly shorter than those in the normal saline control group (p0.05), while the swimming time and distance of the combined drug treatment group were shorter (p0.05). Compared with the saline group, the number of crossing platform in the combined drug treatment group was significantly increased (p0.01), and the number of crossing platform in the single drug treatment group was also significantly increased (p0.05); compared with the combined drug treatment group, VPA treatment group and liq treatment group were significantly reduced (p0.05), E2 treatment group was significantly reduced (p0.01). uterine morphology and weight statistics of mice in each group showed that: The uterus morphology of the mice in the treatment group increased in different degrees. the results of statistical analysis showed that the weight of uterus in VPA group, liq group and control group had no significant change (p0.05); the weight of uterus in E2 group, VPA + E2 group, VPA + liq group and control group increased significantly (p0.01). The results of protein level experiment: (1) compared with the normal saline and estrogen receptor alpha group, the content of ER alpha protein in the cerebral cortex increased significantly in the combined drug treatment group (p0.01), and there was no significant difference in the single drug treatment group (p0.05). Compared with the control group, the level of ER-alpha protein in the cerebral hippocampus did not change significantly in the VPA treatment group (p0.05), but increased significantly in the other drug treatment groups (p0.05). in the comparison of ER-beta content, compared with the normal saline control group, the content of ER-beta in the cerebral cortex and hippocampus of the other drug treatment groups increased significantly (p0.05). (2) GSK-3 beta activity test: compared with the normal saline control group, there was no significant difference in the levels of GSK-3 beta total protein in the cerebral cortex and hippocampus of mice treated with other drugs alone (p0.05), and there was no significant difference in GSK-3 beta total protein between the combined drug treatment group and the normal saline control group (p0.05). The total protein level decreased significantly (p0.05), but there was no significant difference in the total protein level of GSK-3 beta in the hippocampus (p0.05). the phosphorylation level of the ninth locus marked the activity of GSK-3 beta. compared with the normal saline control group, the levels of pser9-gsk-3 beta in the VPA group, E2 group and liq group had no significant difference (p0.05), and the combined drug treatment group. The levels of pser9-gsk-3 beta in the hippocampus of each group were significantly higher than those in the normal saline group (p0.05). The levels of pser9-gsk-3 beta in the other drug treatment groups were significantly higher than those in the control group (p0.05), and the levels of pser9-gsk-3 beta in the combined drug treatment group were also significantly higher than those in the single drug treatment group (p0.05). Conclusion: 1. After ovariectomy, the estrogen level in the systemic circulation of AD mice decreased with age, and the uterine morphology gradually atrophied with age. 2. After ovariectomy, the spatial learning and memory ability of AD mice gradually decreased with age. 3. After ovariectomy, the deposition of A beta in the brain and the formation of senile plaque of AD mice increased, which may be related to ovariectomy. The mechanism may be related to the abnormal GSK-3 beta activity caused by the decrease of ER beta content. 4. Drug treatment significantly improved the depressed and anxious behavior of ovariectomized AD mice, alleviated the decline of their spatial learning and memory ability, and the effect of combined treatment was significantly better than that of single drug treatment. 5. Drug treatment significantly increased the cortex and sea of ovariectomized AD mice. The content of estrogen receptor in horse area was significantly increased. 6. Drug treatment significantly increased the content of P ser9-GSK-3 beta in cerebral cortex and hippocampus of ovariectomized AD mice, and inhibited the activity of GSK-3 beta, and the effect of combined treatment was more significant.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16
【参考文献】
相关期刊论文 前4条
1 刘刚;廖娥;钟国超;王凌f^;周泽芬;贺桂琼;;不同年龄段雌性APP/PS1双转基因小鼠脑内病理特征的动态变化[J];重庆医科大学学报;2015年01期
2 商秀丽;赵久晗;曹云鹏;薛一雪;;雌二醇对阿尔茨海默病大鼠学习记忆能力及长时程增强的影响(英文)[J];Neuroscience Bulletin;2010年02期
3 杨华;屈秋民;郭峰;韩建峰;武成斌;;雌激素在阿尔茨海默病患者的改变及其意义[J];中国神经精神疾病杂志;2007年03期
4 常艳,傅一山,朱心强;小鼠卵巢切除术入路的改良[J];上海实验动物科学;1999年04期
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